Method to detect prostate cancer in a sample

ABSTRACT

The present invention relates to prostate cancer. More specifically, the present invention relates to a method to detect prostate cancer in a patient sample by detecting the RNA encoded by the gene PCA3. More particularly the present invention relates to a method for determining a predisposition, or presence of prostate cancer in a patient comprising: (a) contacting a biological sample of a patient with at least one oligonucleotide that hybridizes to a PCA3 polynucleotide; (b) detecting in the biological sample an amount of PCA3 and second prostate specific polynucleotides; and (c) comparing the amount of PCA3 polynucleotide that hybridizes to the oligonucleotide to a predetermined cut off value, and therefrom determining the presence or absence of prostate cancer in the biological sample. The present invention further relates to diagnostic kits for the detection of prostate cancer or the risk of developing same in a patient comprising: (a) at least one container means having disposed therein at least one oligonucleotide probe or primer that hybridizes to one a PCA3 nucleic acid or complement thereof; (b) at least one oligonucleotide probe or primer that hybridizes with a second prostate specific nucleic acid or complement thereof; and (c) reagents enabling a detection of PCA3 and of the second prostate specific nucleic acid when PCA3 or second prostate-specific nucleic acid sequence is present.

FIELD OF THE INVENTION

The present invention relates to prostate cancer. More specifically, thepresent invention relates to a method to detect prostate cancer in apatient sample by detecting an RNA encoded by the prostate cancerantigen PCA3 gene.

BACKGROUND OF THE INVENTION

Over the last decade, cancer of the prostate has become the mostcommonly diagnosed malignancy among men and the second leading cause ofmale cancer deaths in the western population, following lung cancer.

Early detection and treatment of prostate cancer before it has spreadfrom the prostate gland, reduces the mortality of the disease. This isparticularly true for younger men who are at greater risk of dying fromthis pernicious but slowly growing malignancy. This realization hasprompted increasing efforts for early diagnosis and treatment. Indeed,the American Cancer Society and the American Urological Associationrecommend that male population at large undergo annual screening forprostate cancer beginning at age 50. The recommended age for screeningis lowered to 40 for men giving a family history of prostate cancer orother risk factors.

With this increasing focus on prostate cancer screening, more men thanever before are being routinely tested for prostate cancer. Notsurprisingly, this practice has increased early detection of onset ofthe disease, as reflected by an apparent increase in the incidence ofprostate cancer and decrease in the apparent average age of diagnosis.The clinical hope is that earlier detection of prostate cancer before itmetastasizes will reduce the overall mortality rate. Healthcare payerslook for early screening and detection to translate into a reduction inthe healthcare burden, as early treatment can be less radical, moresuccessful and therefore provided at a lower cost per treated patient.The key to accomplishing this goal remains providing better differentialdiagnostic tools.

Screening for prostate cancer now involves both palpation of theprostate by digital rectal examination (DRE) and assay of plasma levelsof prostate specific antigen (PSA/hK3/hKLK3). PSA is a serine proteaseproduced by the prostatic epithelium that is normally secreted in theseminal fluid to liquefy it. Disruption of the anatomic integrity of theprostate gland can compromise the cellular barriers that normallyrestrict PSA to within the duct system of the prostate, allowing it todisperse into blood or urine. A number of conditions can result inleakage of PSA into the blood. They include inflammation of theprostate, urinary retention, prostatic infection, benign prostatichyperplasia (BPH), and prostate cancer. Physical manipulation of theprostate can also increase serum PSA levels, but a mild stimulus, suchas digital rectal examination (DRE), does not normally increase serumPSA. It is therefore not surprising that screening of serum PSA as anindicator of prostate cancer is not absolutely predictive.

Despite the fact that measure of blood PSA levels can be the result froma variety of different causes, it is nonetheless the basis for primaryscreening for prostate cancer. Measurement of total PSA (tPSA) as adiagnostic assay to predict prostate cancer has been in use since 1991.Levels of 4 ng/ml or greater in blood serum are considered abnormal andpredictive of prostate cancer. However, the sensitivity of such elevatedtPSA levels is only 79%; thus leaving 21% of patients with prostatecancer undetected. The specificity for all tPSA values of 4 ng/ml orgreater is very poor. In addition, estimates of specificity for tPSAlevels >4.0 ng/ml are reported to be in the range of 20% to 59%,averaging around 33%. The vast majority of false positives areultimately shown to be benign prostatic hyperplasia (BPH). Thespecificity is lowest for modestly elevated tPSA, in the low so-calledgray zone of 4 to 10 ng/ml. This low level of specificity results inadditional more invasive and costly diagnostic procedures, such astransrectal ultrasounds and prostate biopsies. Such tests whenunnecessary are also very traumatic for the patient. The psychologicalimpact of being diagnosed as positive until proven as a false positiveshould not be understated either.

Because of the shortcomings of tPSA, research has been focused onattempting to develop PSA derivatives to increase the sensitivity andspecificity of this general diagnostic approach.

One modification is free PSA (fPSA), which was FDA approved in 1998. PSAin serum can be found either in an unbound form or complexed withcirculating protease inhibitors, most commonly with alpha-1-antitrypsin(ACT). Clinicians have shown that the proportion of PSA bound to ACT wassignificantly higher in men with prostate cancer than in unaffected menor those with BPH. As a guideline, if 25% orless of total PSA is free,this is an indicator of possible prostate cancer. The fPSA assay wasapproved for use in men with tPSA's for 4 to 10 ng/ml. Thus, the fPSAassay was positioned to improve the specificity over that of tPSA alone.However, the predictivity of the fPSA test is not as good in people withreally low or really high tPSA levels. Very low tPSA, regardless ofmeasured fPSA, is predictive of not having cancer, while the converse istrue with very high tPSA levels. The diagnostic usefulness of fPSA isrelatively limited as it can be associated with either BPH or prostatecancer. The use of fPSA in combination with tPSA has been shown toreduce the number of unnecessary biopsies by about 20%.

Clearly, prostate biopsy is the gold standard for confirming prostatecancer. However, even a biopsy is not always 100% certain. The standardis the sextant biopsy where tissue sample collection is guided bytransrectal ultrasound. Often six samples are not enough to detect thecancer and either a second biopsy procedure or more than six samples arerequired.

Despite the improvements in prostate cancer screening over the last tenyears, there remains a large unmet need in diagnostic sensitivity andspecificity, even when these tools are used in combination. Couplingthis need with the large incidence of prostate cancer and the importancefor early, accurate detection, the potential usefulness for a truedifferential diagnostic tool is very significant.

A new prostate cancer marker, PCA3, was discovered a few years ago bydifferential display analysis intended to highlight genes associatedwith prostate cancer development. PCA3 is located on chromosome 9 andcomposed of four exons. It encodes at least four different transcriptswhich are generated by alternative splicing and polyadenylation. ByRT-PCR analysis, PCA3 expression was found to be limited to the prostateand absent in all other tissues tested, including testis, ovary, breastand bladder. Northern blot analysis showed that PCA3 is highly expressedin the vast majority of prostate cancers examined (47 out of 50) whereasno or very low expression is detected in BPH or normal prostate cellsfrom the same patients [Cancer Res 1999 December 1;59(23):5975-9].Moreover, a recent study comparing the clinical performance of RNAtelomerase RT and RNA PCA3 detection in the case of prostate cancershowed that the PCA3 gene can be considered as a better marker (CancerRes 2002 May 1;62(9):2695-8).

The PCA3 gene is composed of 4 exons (e1-e4) and 3 introns (i1-i3).While PCA3 appears to be recognized as the best prostate-cancer markerever identified, this specificity has been contested in the literature.For example, Gandini et al. 2003, claim that the prostate-specificexpression of PCA3 is restricted to that of exon 4 of the PCA3 gene.However, the applicants have shown in a recent patent application thatthis is not the case (Patent application CA 2,432,365).

In view of the fact that advanced prostate cancer remains a lifethreatening disease reaching a very significant proportion of the malepopulation, there remains a need to provide the most specific,selective, and rapid prostate cancer detection methods and kits.

The present invention seeks to meet these and other needs.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention relates to diagnostic methods and kits to detectprostate cancer, which are more specific and selective than the methodsand kits of the prior art.

The present invention relates to a method to detect prostate cancer in apatient and especially from a urine sample thereof by detecting the RNAencoded by the PCA3 gene.

The invention further relates to a method of diagnosing the presence orpredisposition to develop prostate cancer in a patient. Also disclosedis a method for monitoring the progression of prostate cancer in apatient.

In one particular embodiment, the present invention relates to a methodto detect prostate cancer in urine samples by detecting the presence ofRNA encoded by the PCA3 gene. In one embodiment, RNA encoded by the PCA3gene is detected using an amplification method, which simultaneouslyamplifies a second prostate-specific nucleic acid sequence alsocontained in the sample.

In one further particular embodiment of the present invention, theamplified second prostate specific marker is selected from the groupconsisting of PSA, human kallikrein 2 (hK2/KLK2), prostate specificmembrane antigen (PSMA), transglutaminase 4, acid phosphatase or PCGEM1RNA.

In another embodiment of the present invention, the RNA is detectedusing an RNA amplification method. In a further embodiment, the RNAamplification method is coupled to real-time detection of the amplifiedproducts using fluorescence specific probes. In yet a furtherembodiment, the amplification method is PCR. In an additional embodimentthe PCR is real-time PCR or a related method enabling detection inreal-time of the amplified products.

In a related embodiment RNA encoded by the PCA3 gene is detected in anucleic acid extract by an in vitro RNA amplification method namedNucleic Acid Based Amplification (NASBA). Of course other RNAamplification methods are known and the instant methods and kits aretherefore not limited to NASBA. Non-limiting examples of such RNAamplification methods include transcriptase mediated amplification(TMA), strand displacement amplification (SDA) and ligase chain reaction(LCR).

In a further embodiment, the amplified products are detected in ahomogenous phase using a fluorescent probe. In one embodiment, theBeacon approach is used. In another embodiment, the product is detectedon solid phase using fluorescent or colorimetric method. It should thusbe understood that numerous fluorescent, calorimetric or enzymaticmethods can be used in accordance with the present invention to detectand/or quantify RNAs. Other types of labelled probes and primers orother types of detection methods may also be used in the presentinvention (e.g., hybridization assays such as Northern blots, dot blotsor slot blots and radiolabelled probes and primers).

In one embodiment, the RNA encoded by the PCA3 gene is obtained from acell contained in a voided urine sample from the patient.

In another embodiment, the urine sample is obtained after an attentivedigital rectal examination (DRE). Of course, it should be understoodthat the present methods and kits could also be used on a urine sampleobtained without DRE, or on other types of samples such as sperm ormixed urine and sperm (e.g., first urine sample following ejaculation),provided that the amplification method and/or detection method issensitive enough to detect the targeted markers (PCA3 and secondmarker). Experiments showed that the methods and kits of the presentinvention can also be performed with these types of samples. Othersamples that can be used include blood or serum.

In one embodiment, the cells collected from the urine sample areharvested and a total nucleic acid extraction is carried out. In oneparticular embodiment, total nucleic acid extraction is carried outusing a solid phase band method on silica beads as described by BOOM etal., (1990, J. Clin. Microbiol. 28: 495-503). In another embodiment, thenucleic acids are purified using another target capture method (seebelow). Of course, it should be understood that numerous nucleic acidextraction and purification methods exist and thus, that other methodscould be used in accordance with the present invention. Non-limitingexamples include a phenol/chloroform extraction method and targetcapture purification method (see below). Other such methods aredescribed in herein referenced textbooks. It should also be recognizedthat numerous means to stabilize or protect the prostate cells containedin the urine sample or other sample, as well as to stabilize or protectthe RNA present in these cells are well known in the art.

In another embodiment, the methods of the present invention are carriedout using a crude, unpurified, or semi-purified sample.

In one particular embodiment, the present invention also relates to aprostate cancer diagnostic kit for detecting the presence of PCA3nucleic acid in a sample. Such kit generally comprises a first containermeans having disposed therein at least one oligonucleotide probe and/orprimer that hybridizes to a PCA3 nucleic acid (e.g. PCA3 RNA) and asecond container means containing at least one other oligonucleotideprimer and/or probe that hybridizes to the above-mentioned secondprostate-specific sequence. In another embodiment, a third containermeans contains a probe which specifically hybridizes to the PCA3amplification product. In a preferred embodiment, the kit furtherincludes other containers comprising additional components such as aadditional oligonucleotide or primer and/or one or more of thefollowing: buffers, reagents to be used in the assay (e.g. washreagents, polymerases, internal controls (IC) or else) and reagentscapable of detecting the presence of bound nucleic acidprobe(s)/primer(s). Of course numerous embodiments of the kits of thepresent invention are possible. For example, the different containermeans can be divided in amplifying reagents and detection reagents. Inone such an embodiment, a first container means contains amplificationor hybridization reagents specific for the target nucleic acids of thepresent invention (e.g., PCA 3, second prostate specific and internalcontrol nucleic acids) and the second container means contains detectionreagents. Alternatively, the detection reagents and amplificationreagents can be contained in the same container mean.

The present invention in addition relates to a prostate cancerdiagnostic kit for detecting the presence of PCA3 nucleic acid in asample. Such kit generally comprises a first container means havingdisposed therein at least one oligonucleotide probe and/or primer thathybridizes to a PCA3 mRNA and a second container means containing atleast one other oligonucleotide primer and/or probe that hybridizes tothe mRNA of the second prostate-specific sequence. In anotherembodiment, a third container means contains a probe which specificallyhybridizes to the PCA3 amplification product. In a yet anotherembodiment a fourth container means contains a probe which specificallyhybridizes to the second prostate specific mRNA. In a preferredembodiment, the kit further includes other containers comprisingadditional components such as a additional oligonucleotide or primer(e.g., for internal control) and/or one or more of the following:buffers, reagents to be used in the assay (e.g. wash reagents,polymerases, internal control nucleic acid or cells or else) andreagents capable of detecting the presence of bound nucleic acidprobe(s)/primer(s). Of course the separation or assembly of reagents insame or different container means is dictated by the types ofextraction, amplification or hybridization methods, and detectionmethods used as well as other parameters including stability, need forpreservation etc.

Multiple methods and kits are encompassed by the present invention. Forexample, the detection and or amplification of the PCA3 nucleic acidsequence does not need to be identical to that of the second prostatespecific polynucleotide or other targeted sequences. Thus for example amethod or kit which would be RNA based for PCA3 could be DNA based forthe second prostate marker or for other targeted sequences.

It should be understood by a person of ordinary skill that numerousstatistical methods can be used in the context of the present inventionto determine if the test is positive or negative. The decisional treeused is only one non-limiting example of such a statistical method.

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Commonlyunderstood definitions of molecular biology terms can be found forexample in Dictionary of Microbiology and Molecular Biology, 2nd ed.(Singleton et al., 1994, John Wiley & Sons, New York, N.Y.) or TheHarper Collins Dictionary of Biology (Hale & Marham, 1991, HarperPerennial, New York, N.Y.), Rieger et al., Glossary of genetics:Classical and molecular, 5^(th) edition, Springer-Verlag, N.Y., 1991;Alberts et al., Molecular Biology of the Cell, 4^(th) edition, Garlandscience, New-York, 2002; and, Lewin, Genes VII, Oxford University Press,New-York, 2000. Generally, the procedures of molecular biology methodsand the like are common methods used in the art. Such standardtechniques can be found in reference manuals such as for exampleSambrook et al. (2000, Molecular Cloning—A Laboratory Manual, ThirdEdition, Cold Spring Harbor Laboratories); and Ausubel et al. (1994,Current Protocols in Molecular Biology, John Wiley & Sons, New-York).

In the present description, a number of terms are extensively utilized.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

DEFINITIONS

Nucleotide sequences are presented herein by single strand, in the 5′ to3′ direction, from left to right, using the one letter nucleotidesymbols as commonly used in the art and in accordance with therecommendations of the IUPAC-IUB Biochemical Nomenclature Commission.

The present description refers to a number of routinely used recombinantDNA (rDNA) technology terms. Nevertheless, definitions of selectedexamples of such rDNA terms are provided for clarity and consistency.

As used herein, “nucleic acid molecule” or “polynucleotides”, refers toa polymer of nucleotides. Non-limiting examples thereof include DNA(e.g. genomic DNA, cDNA), RNA molecules (e.g. mRNA) and chimerasthereof. The nucleic acid molecule can be obtained by cloning techniquesor synthesized. DNA can be double-stranded or single-stranded (codingstrand or non-coding strand [antisense]). Conventional ribonucleic acid(RNA) and deoxyribonucleic acid (DNA) are included in the term “nucleicacid” and polynucleotides as are analogs thereof. A nucleic acidbackbone may comprise a variety of linkages known in the art, includingone or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds(referred to as “peptide nucleic acids” (PNA); Hydig-Hielsen et al., PCTInt'l Pub. No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages or combinations thereof. Sugar moieties ofthe nucleic acid may be ribose or deoxyribose, or similar compoundshaving known substitutions, e.g., 2′ methoxy substitutions (containing a2′-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2′halide substitutions. Nitrogenous bases may be conventional bases (A, G,C, T, U), known analogs thereof (e.g., inosine or others; see TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992), or known derivatives of purine or pyrimidine bases (see, Cook,PCT Int'l Pub. No. WO 93/13121) or “abasic” residues in which thebackbone includes no nitrogenous base for one or more residues (Arnoldet al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases and linkages, as found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more base analogs). Theterminology “PCA3 nucleic acid” or “PCA3 polynucleotides” refers to anative PCA3 nucleic acid sequence. In one embodiment, the PCA3 nucleicacid has the sequence has set forth in SEQ ID NOs 9,10 and 13. Inanother embodiment, the PCA3 nucleic acid encodes a PCA3 protein. In afurther embodiment, the PCA3 nucleic acid is a non-coding nucleic acidsequence. In yet a further embodiment, the PCA3 sequence which istargeted by the PCA3 sequences encompassed by the present invention, isa natural PCA3 sequence found in a patient sample.

The term “recombinant DNA” as known in the art refers to a DNA moleculeresulting from the joining of DNA segments. This is often referred to asgenetic engineering. The same is true for “recombinant nucleic acid”.

The term “DNA segment” is used herein, to refer to a DNA moleculecomprising a linear stretch or sequence of nucleotides. This sequencewhen read in accordance with the genetic code (e.g., an open readingframe or ORF), can encode a linear stretch or sequence of amino acidswhich can be referred to as a polypeptide, protein, protein fragment andthe like.

The terminology “amplification pair” or “primer pair” refers herein to apair of oligonucleotides (oligos) of the present invention, which areselected to be used together in amplifying a selected nucleic acidsequence by one of a number of types of amplification processes.

“Amplification” refers to any known in vitro procedure for obtainingmultiple copies (“amplicons”) of a target nucleic acid sequence or itscomplement or fragments thereof. In vitro amplification refers toproduction of an amplified nucleic acid that may contain less than thecomplete target region sequence or its complement. Known in vitroamplification methods include, e.g., transcription-mediatedamplification, replicase-mediated amplification, polymerase chainreaction (PCR) amplification, ligase chain reaction (LCR) amplificationand strand-displacement amplification (SDA). Replicase-mediatedamplification uses self-replicating RNA molecules, and a replicase suchas Qβ-replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600). PCRamplification is well known and uses DNA polymerase, primers and thermalcycling to synthesize multiple copies of the two complementary strandsof DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159). LCR amplification uses at least four separateoligonucleotides to amplify a target and its complementary strand byusing multiple cycles of hybridization, ligation, and denaturation(e.g., EP Pat. App. Pub. No.0 320 308). SDA is a method in which aprimer contains a recognition site for a restriction endonuclease thatpermits the endonuclease to nick one strand of a hemimodified DNA duplexthat includes the target sequence, followed by amplification in a seriesof primer extension and strand displacement steps (e.g., Walker et al.,U.S. Pat. No. 5,422,252). Another known strand-displacementamplification method does not require endonuclease nicking (Dattaguptaet al., U.S. Pat. No. 6,087,133). Transcription-mediated amplificationis used in the present invention. Those skilled in the art willunderstand that the oligonucleotide primer sequences of the presentinvention may be readily used in any in vitro amplification method basedon primer extension by a polymerase. (see generally Kwoh et al., 1990,Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad.Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202;Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al.,2000, Molecular Cloning—A Laboratory Manual, Third Edition, CSHLaboratories). As commonly known in the art, the oligos are designed tobind to a complementary sequence under selected conditions.

As used herein, the term “physiologically relevant” is meant to describeinteractions that can modulate a function which is physiologicallyrelevant. In the present invention, encompassed for example thetranscription of a gene in its natural setting. Of course a binding of aprotein to PCA3 may also be considered as a physiologically relevantfunction if this binding occur in a natural setting.

The term “DNA” molecule or sequence (as well as sometimes the term“oligonucleotide”) refers to a molecule comprised generally of thedeoxyribonucleotides adenine (A), guanine (G), thymine (T) and/orcytosine (C). In “RNA”, T is replaced by uracil (U). As used herein,particular DNA or RNA sequences may be described according to the normalconvention of giving only the sequence in the 5′ to 3′ direction.

Agarose Gel Electrophoresis. The most commonly used technique (thoughnot the only one) for fractionating double stranded DNA is agarose gelelectrophoresis. The principle of this method is that DNA moleculesmigrate through the gel as though it were a sieve that retards themovement of the largest molecules to the greatest extent and themovement of the smallest molecules to the least extent. Note that thesmaller the DNA fragment, the greater the mobility under electrophoresisin the agarose gel.

The DNA fragments fractionated by agarose gel electrophoresis can bevisualized directly by a staining procedure if the numberof fragmentsincluded in the pattern is small. In ordertovisualize a small subset ofthese fragments, a methodology referred to as a hybridization procedure(e.g., Southern hybridization) can be applied.

“Nucleic acid hybridization” refers generally to the hybridization oftwo single-stranded nucleic acid molecules having complementary basesequences, which under appropriate conditions will form athermodynamically favored double-stranded structure. Examples ofhybridization conditions can be found in the two laboratory manualsreferred above (Sambrook et al., 2000, supra and Ausubel et al., 1994,supra) and are commonly known in the art. In the case of a hybridizationto a nitrocellulose filter (or other such support like nylon), as forexample in the well known Southern blotting procedure, a nitrocellulosefilter can be incubated overnight at 65° C. with a labeled probe in asolution containing high salt (6×SSC or 5×SSPE), 5× Denhardt's solution,0.5% SDS, and 100 μg/ml denatured carrier DNA (e.g. salmon sperm DNA).The non-specifically binding probe can then be washed off the filter byseveral washes in 0.2×SSC/0.1% SDS at a temperature which is selected inview of the desired stringency: room temperature (low stringency), 42°C. (moderate stringency) or 65° C. (high stringency). The salt and SDSconcentration of the washing solutions may also be adjusted toaccommodate for the desired stringency. The selected temperature andsalt concentration is based on the melting temperature (Tm) of the DNAhybrid. Of course, RNA-DNA hybrids can also be formed and detected. Insuch cases, the conditions of hybridization and washing can be adaptedaccording to well known methods by the person of ordinary skill.Stringent conditions will be preferably used (Sambrook et al., 2000,supra). Other protocols or commercially available hybridization kits(e.g., ExpressHyb™ from BD Biosciences Clonetech) using differentannealing and washing solutions can also be used as well known in theart.

A “probe” is meant to include a nucleic acid oligomer that hybridizesspecifically to a target sequence in a nucleic acid or its complement,under conditions that promote hybridization, thereby allowing detectionof the target sequence or its amplified nucleic acid. Detection mayeither be direct (i.e, resulting from a probe hybridizing directly tothe target or amplified sequence) or indirect (i.e., resulting from aprobe hybridizing to an intermediate molecular structure that links theprobe to the target or amplified sequence). A probe's “target” generallyrefers to a sequence within an amplified nucleic acid sequence (i.e, asubset of the amplified sequence) that hybridizes specifically to atleast a portion of the probe sequence by standard hydrogen bonding or“base pairing.” Sequences that are “sufficiently complementary” allowstable hybridization of a probe sequence to a target sequence, even ifthe two sequences are not completely complementary. A probe may belabeled or unlabeled.

By “sufficiently complementary” is meant a contiguous nucleic acid basesequence that is capable of hybridizing to another sequence by hydrogenbonding between a series of complementary bases. Complementary basesequences may be complementary at each position in sequence by usingstandard base pairing (e.g., G:C, A:T or A:U pairing) or may contain oneor more residues (including abasic residues) that are not complementaryby using standard base pairing, but which allow the entire sequence tospecifically hybridize with another base sequence in appropriatehybridization conditions. Contiguous bases of an oligomer are preferablyat least about 80% (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90%complementary to the sequence to which the oligomer specificallyhybridizes. Appropriate hybridization conditions are well known to thoseskilled in the art, can be predicted readily based on sequencecomposition and conditions, or can be determined empirically by usingroutine testing (see Sambrook et al., Molecular Cloning, A LaboratoiyManual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57, particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

Nucleic acid sequences may be detected by using hybridization with acomplementary sequence (e.g., oligonucleotide probes) (see U.S. Pat. No.5,503,980 (Cantor), U.S. Pat. No. 5,202,231 (Drmanac et al.), U.S. Pat.No. 5,149,625 (Church et al.), U.S. Pat. No. 5,112,736 (Caldwell etal.), U.S. Pat. No. 5,068,176 (Vijg et al.), and U.S. Pat. No. 5,002,867(Macevicz)). Hybridization detection methods may use an array of probes(e.g., on a DNA chip) to provide sequence information about the targetnucleic acid which selectively hybridizes to an exactly complementaryprobe sequence in a set of four related probe sequences that differ onenucleotide (see U.S. Pat. Nos. 5,837,832 and 5,861,242 (Chee et al.)).

A detection step may use any of a variety of known methods to detect thepresence of nucleic acid by hybridization to a probe oligonucleotide.One specific example of a detection step uses a homogeneous detectionmethod such as described in detail previously in Arnold et al. ClinicalChemistry 35:1588-1594 (1989), and U.S. Pat. No. 5,658,737 (Nelson etal.), and U.S. Pat. Nos. 5,118,801 and 5,312,728 (Lizardi et al.).

The types of detection methods in which probes can be used includeSouthern blots (DNA detection), dot or slot blots (DNA, RNA), andNorthern blots (RNA detection). Labeled proteins could also be used todetect a particular nucleic acid sequence to which it binds (e.g proteindetection by far western technology: Guichet et al., 1997, Nature385(6616): 548-552; and Schwartz et al., 2001, EMBO 20(3): 510-519).Other detection methods include kits containing reagents of the presentinvention on a dipstick setup and the like. Of course, it might bepreferable to use a detection method which is amenable to automation. Anon-limiting example thereof includes a chip or other support comprisingone or more (e.g. an array) of different probes.

A “label” refers to a molecular moiety or compound that can be detectedor can lead to a detectable signal. A label is joined, directly orindirectly, to a nucleic acid probe or the nucleic acid to be detected(e.g., an amplified sequence). Direct labeling can occur through bondsor interactions that link the label to the nucleic acid (e.g., covalentbonds or non-covalent interactions), whereas indirect labeling can occurthrough use a “linker” or bridging moiety, such as additionaloligonucleotide(s), which is either directly or indirectly labeled.Bridging moieties may amplify a detectable signal. Labels can includeany detectable moiety (e.g., a radionuclide, ligand such as biotin oravidin, enzyme or enzyme substrate, reactive group, chromophore such asa dye or colored particle, luminescent compound including abioluminescent, phosphorescent or chemiluminescent compound, andfluorescent compound). Preferably, the label on a labeled probe isdetectable in a homogeneous assay system, i.e., in a mixture, the boundlabel exhibits a detectable change compared to an unbound label.

Other methods of labeling nucleic acids are known whereby a label isattached to a nucleic acid strand as it is fragmented, which is usefulfor labeling nucleic acids to be detected by hybridization to an arrayof immobilized DNA probes (e.g., see PCT No. PCT/IB99/02073).

A “homogeneous detectable label” refers to a label whose presence can bedetected in a homogeneous fashion based upon whether the labeled probeis hybridized to a target sequence. A homogeneous detectable label canbe detected without physically removing hybridized from unhybridizedforms of the labeled probe. Homogeneous detectable labels and methods ofdetecting them have been described in detail elsewhere (e.g., see U.S.Pat. Nos. 5,283,174, 5,656,207 and 5,658,737).

As used herein, “oligonucleotides” or “oligos” define a molecule havingtwo or more nucleotides (ribo or deoxyribonucleotides). The size of theoligo will be dictated by the particular situation and ultimately on theparticular use thereof and adapted accordingly by the person of ordinaryskill. An oligonucleotide can be synthesized chemically or derived bycloning according to well known methods. While they are usually in asingle-stranded form, they can be in a double-stranded form and evencontain a “regulatory region”. They can contain natural rare orsynthetic nucleotides. They can be designed to enhance a chosen criterialike stability for example.

As used herein, a “primer” defines an oligonucleotide which is capableof annealing to a target sequence, thereby creating a double strandedregion which can serve as an initiation point for nucleic acid synthesisunder suitable conditions. Primers can be, for example, designed to bespecific for certain alleles so as to be used in an allele-specificamplification system. For example, a primer can be designed so as to becomplementary to a short PCA3 RNA which is associated with a malignantstate of the prostate, whereas a long PCA3 RNA is associated with anon-malignant state (benign) thereof (PCT/CA00/01154 published under No.WO 01/23550). The primer's 5′ region may be non-complementary to thetarget nucleic acid sequence and include additional bases, such as apromoter sequence (which is referred to as a “promoter primer”). Thoseskilled in the art will appreciate that any oligomer that can functionas a primer can be modified to include a 5′ promoter sequence, and thusfunction as a promoter primer. Similarly, any promoter primer can serveas a primer, independent of its functional promoter sequence. Of coursethe design of a primer from a known nucleic acid sequence is well knownin the art. As for the oligos, it can comprise a number of types ofdifferent nucleotides.

Transcription-associated amplification. Amplifying a target nucleic acidsequence by using at least two primers can be accomplished using avariety of known nucleic acid amplification methods, but preferably usesa transcription-associated amplification reaction that is substantiallyisothermal. By using such an in vitro amplification method, many strandsof nucleic acid are produced from a single copy of target nucleic acid,thus permitting detection of the target in the sample by specificallybinding the amplified sequences to one or more detection probes.Transcription-associated amplification methods have been described indetail elsewhere (e.g., U.S. Pat. Nos. 5,399,491 and 5,554,516).Briefly, transcription-associated amplification uses two types ofprimers (one being a promoter primer because it contains a promotersequence for an RNA polymerase), two enzyme activities (a reversetranscriptase (RT) and an RNA polymerase), substrates(deoxyribonucleoside triphosphates, ribonucleoside triphosphates) andappropriate salts and buffers in solution to produce multiple RNAtranscripts from a nucleic acid template. Initially, a promoter primerhybridizes specifically to a target sequence (e.g., RNA) and reversetranscriptase creates a first complementary DNA strand (cDNA) byextension from the 3′ end of the promoter primer. The cDNA is madeavailable for hybridization with the second primer by any of a varietyof methods, such as, by denaturing the target-cDNA duplex or using RNaseH activity supplied by the RT that degrades RNA in a DNA:RNA duplex. Asecond primer binds to the cDNA and a new strand of DNA is synthesizedfrom the end of the second primer using the RT activity to create adouble-stranded DNA (dsDNA) having a functional promoter sequence at oneend. An RNA polymerase binds to the dsDNA promoter sequence andtranscription produces multiple transcripts (“amplicons”). Amplicons areused in subsequent steps or cycles of the transcription-associatedamplification process by serving as a new template for replication, thusgenerating many copies of amplified nucleic acid (i.e., about 100 to3,000 copies of RNA are synthesized from each template).

NASBA. Nucleic Acid Sequence Based Amplification (NASBA) can be carriedout in accordance with known techniques (Malek et al. Methods Mol Biol,28:253-260). In an embodiment, the NASBA amplification starts with theannealing of an antisense primer P1 (containing the T7 RNA polymerasepromoter) to the mRNA target. Reverse transcriptase (RTase) thensynthesizes a complementary DNA strand. The double stranded DNA/RNAhybrid is recognized by RNase H that digests the RNA strand, leaving asingle-stranded DNA molecule to which the sense primer P2 can bind. P2serves as an anchor to the RTase that synthesizes a second DNA strand.The resulting double-stranded DNA has a functional T7 RNA polymerasepromoter recognized by the respective enzyme. The NASBA reaction canthen enter in the phase of cyclic amplification comprising six steps:(1) Synthesis of short antisense single-stranded RNA molecules (10¹ to10³ copies per DNA template) by the T7 RNA polymerase; (2) annealing ofprimer P2 to these RNA molecules; (3) synthesis of a complementary DNAstrand by RTase; (4) digestion of the RNA strand in the DNA/RNA hybrid;(5) annealing of primer P1 to the single-stranded DNA; and (6)generation of double stranded DNA molecules by RTase. Because the NASBAreaction is isothermal (41° C.), specific amplification of ssRNA ispossible if denaturation of dsDNA is prevented in the sample preparationprocedure. It is thus possible to pick up RNA in a dsDNA backgroundwithout getting false positive results caused by genomic dsDNA.

Polymerase chain reaction (PCR). Polymerase chain reaction can becarried out in accordance with known techniques. See, e.g., U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the disclosures ofall three U.S. Patent are incorporated herein by reference). In general,PCR involves, a treatment of a nucleic acid sample (e.g., in thepresence of a heat stable DNA polymerase) under hybridizing conditions,with one oligonucleotide primer for each strand of the specific sequenceto be detected. An extension product of each primer which is synthesizedis complementary to each of the two nucleic acid strands, with theprimers sufficiently complementary to each strand of the specificsequence to hybridize therewith. The extension product synthesized fromeach primer can also serve as a template for further synthesis ofextension products using the same primers. Following a sufficient numberof rounds of synthesis of extension products, the sample is analyzed toassess whether the sequence or sequences to be detected are present.Detection of the amplified sequence may be carried out by visualizationfollowing EtBr staining of the DNA following gel electrophoresis, orusing a detectable label in accordance with known techniques, and thelike. For a review on PCR techniques (see PCR Protocols, A Guide toMethods and Amplifications, Michael et al. Eds, Acad. Press, 1990).

Ligase chain reaction (LCR) can be carried out in accordance with knowntechniques (Weiss, 1991, Science 254:1292). Adaptation of the protocolto meet the desired needs can be carried out by a person of ordinaryskill. Strand displacement amplification (SDA) is also carried out inaccordance with known techniques or adaptations thereof to meet theparticular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).

Target capture. In one embodiment, target capture is included in themethod to increase the concentration or purity of the target nucleicacid before in vitro amplification. Preferably, target capture involvesa relatively simple method of hybridizing and isolating the targetnucleic acid, as described in detail elsewhere (e.g., see U.S. Pat. Nos.6,110,678, 6,280,952, and 6,534,273). Generally speaking, target capturecan be divided in two family, sequence specific and non sequencespecific. In the non-specific method, a reagent (e.g., silica beads) isused to capture non specifically nucleic acids. In the sequence specificmethod an oligonucleotide attached to a solid support is contacted witha mixture containing the target nucleic acid under appropriatehybridization conditions to allow the target nucleic acid to be attachedto the solid support to allow purification of the target from othersample components. Target capture may result from direct hybridizationbetween the target nucleic acid and an oligonucleotide attached to thesolid support, but preferably results from indirect hybridization withan oligonucleotide that forms a hybridization complex that links thetarget nucleic acid to the oligonucleotide on the solid support. Thesolid support is preferably a particle that can be separated from thesolution, more preferably a paramagnetic particle that can be retrievedby applying a magnetic field to the vessel. After separation, the targetnucleic acid linked to the solid support is washed and amplified whenthe target sequence is contacted with appropriate primers, substratesand enzymes in an in vitro amplification reaction.

Generally, capture oligomer sequences include a sequence thatspecifically binds to the target sequence, when the capture method isindeed specific, and a “tail” sequence that links the complex to animmobilized sequence by hybridization. That is, the capture oligomerincludes a sequence that binds specifically to its PCA3 or to anotherprostate specific marker (e.g., PSA, hK2/KLK2, PMSA, transglutaminase 4,acid phosphatase, PCGEM1) target sequence and a covalently attached 3′tail sequence (e.g., a homopolymer complementary to an immobilizedhomopolymer sequence). The tail sequence which is, for example, 5 to 50nucleotides long, hybridizes to the immobilized sequence to link thetarget-containing complex to the solid support and thus purify thehybridized target nucleic acid from other sample components. A captureoligomer may use any backbone linkage, but some embodiments include oneor more 2′-methoxy linkages. Of course, other capture methods are wellknown in the art. The capture method on the cap structure (Edery et al.,1988, gene 74(2): 517-525, U.S. Pat. No. 5,219,989) or the silica basedmethod are two non-limiting examples of capture methods.

An “immobilized probe” or “immobilized nucleic acid” refers to a nucleicacid that joins, directly or indirectly, a capture oligomer to a solidsupport. An immobilized probe is an oligomer joined to a solid supportthat facilitates separation of bound target sequence from unboundmaterial in a sample. Any known solid support may be used, such asmatrices and particles free in solution, made of any known material(e.g., nitrocellulose, nylon, glass, polyacrylate, mixed polymers,polystyrene, silane polypropylene and metal particles, preferablyparamagnetic particles). Preferred supports are monodisperseparamagnetic spheres (i.e., uniform in size±about 5%), thereby providingconsistent results, to which an immobilized probe is stably joineddirectly (e.g., via a direct covalent linkage, chelation, or ionicinteraction), or indirectly (e.g., via one or more linkers), permittinghybridization to another nucleic acid in solution.

The term “allele” defines an alternative form of a gene which occupies agiven locus on a chromosome.

Gene. A DNA sequence generally related but not necessarely related to asingle polypeptide chain or protein, and as used herein includes the 5′and 3′ untranslated regions. The polypeptide can be encoded by afull-length sequence or any portion of the coding sequence, so long asthe functional activity of the protein is retained.

Complementary DNA (cDNA). Recombinant nucleic acid molecules synthesizedby reverse transcription of messenger RNA (“RNA”).

Structural Gene. A DNA sequence that is transcribed into RNA that isthen translated into a sequence of amino acids characteristic of aspecific polypeptide(s).

As commonly known, a “mutation” is a detectable change in the geneticmaterial which can be transmitted to a daughter cell. As well known, amutation can be, for example, a detectable change in one or moredeoxyribonucleotide. For example, nucleotides can be added, deleted,substituted for, inverted, or transposed to a new position. Spontaneousmutations and experimentally induced mutations exist. A mutantpolypeptide can be encoded from this mutant nucleic acid molecule.

As used herein, the term “purified” refers to a molecule (e.g. nucleicacid) having been separated from a component of the composition in whichit was originally present. Thus, for example, a “purified nucleic acid”has been purified to a level not found in nature. A “substantially pure”molecule is a molecule that is lacking in most other components (e.g.,30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free ofcontaminants). By opposition, the term “crude” means molecules that havenot been separated from the components of the original composition inwhich it was present. For the sake of brevity, the units (e.g. 66, 67 .. . 81, 82, . . . 91, 92% . . . ) have not been specifically recited butare considered nevertheless within the scope of the present invention.

As used herein the terminology “prostate specific marker” relates to anymolecule whose presence in the sample indicates that such samplecontains prostate cells (or a marker therefrom). Therefore a “prostatespecific sequence” refers to a nucleic acid or protein sequencespecifically found in prostate cells and usually not in other tissueswhich could “contaminate” a particular sample. For certainty, when aurine sample is used, the second prostate specific marker according tothe present invention does not have to be solely expressed in theprostate. In fact markers which are solely expressed in one organ ortissue is very rare. However, should the second prostate specific markerbe expressed in non-prostate tissue, this non prostate tissue expressionwill not jeopardized the specificity of this second marker provided thatit occurs in cells of tissues or organs which are not normally presentin the urine sample. Thus, when urine is the sample, this secondprostate-specific marker is not normally expressed in other types ofcells (e.g., cells from the urinary tract system) to be found in theurine sample.

Control sample. By the term “control sample” or “normal sample” is meanthere a sample that does not contain a specifically chosen cancer. In aparticular embodiment, the control sample does not contain prostatecancer or is indicative of the absence of prostate cancer. Controlsamples can be obtained from patients/individuals not afflicted withprostate cancer. Other types of control samples may also be used. Forexample, a prostate specific marker can be used as to make sure that thesample contains prostate specific cells (this marker is generallydescribed herein as the second prostate-specific marker). In a relatedaspect, a control reaction may be designed to control the method itself(e.g., The cell extraction, the capture, the amplification reaction ordetection method, number of cells present in the sample, a combinationthereof or any step which could be monitored to positively validate thatthe absence of a signal (e.g., the absence of PCA3 signal) is not theresult of a defect in one ore more of the steps).

Cut-off value. The cut-off value for the predisposition or presence ofprostate cancer is defined from a population of patients withoutprostate cancer as the average signal of PCA3 (or other prostate cancerantigen) polynucleotides, polypeptides or fragments thereof plus nstandard deviations (or average mean signal thereof). Cut off valuesindicative of the presence or predisposition to develop prostate cancermay be the same or alternatively, they may be different values.

Variant. The term “variant” refers herein to a protein or nucleic acidmolecule which is substantially similar in structure and biologicalactivity to the protein or nucleic acid of the present invention, tomaintain at least one of its biological activities. Thus, provided thattwo molecules possess a common activity and can substitute for eachother, they are considered variants as that term is used herein even ifthe composition, or secondary, tertiary or quaternary structure of onemolecule is not identical to that found in the other, or if the aminoacid sequence or nucleotide sequence is not identical.

A “biological sample” or “sample of a patient” is meant to include anytissue or material derived from a living or dead human which may containthe PCA3 target nucleic acid and second prostate specific marker.Samples include, for example, any tissue or material that may containcells specific for the PCA3 target (or second specific marker), such asperipheral blood, plasma or serum, biopsy tissue, gastrointestinaltissue, bone marrow, urine, feces, semen or other body fluids, tissuesor materials, but preferably is a urine sample following digital rectalexamination (or other means which increase the content of prostate cellsin urine). The biological sample may be treated to physically disrupttissue or cell structure, thus releasing intracellular components into asolution which may further contain enzymes, buffers, salts, detergents,and the like which are used to prepare the sample for analysis.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of illustrative embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus, generally described the invention, reference will be madeto the accompanying drawings, showing by way of illustration only anillustrative embodiment thereof and in which:

FIG. 1 shows the PCA3 gene structure and location of oligonucleotidesand probes for in vitro RNA amplification and amplified productdetection. In accordance with one embodiment of the present invention.Panel A. Targeting zone of sense PCA3 primer (SEQ ID NO 4); Panel B.Targeting zone of PCA3 molecular beacon (SEQ ID NO 6); and Panel C.Targeting zone of anti-sense PCA3 primer (SEQ ID NO 3).

FIG. 2 shows a decisional tree used to calculate the positivity of themethod in a patient with total blood PSA below 4 ng/ml.

FIG. 3 shows a decisional tree used to calculate the positivity of themethod in a patient with total blood PSA between 4-10 ng/ml.

FIG. 4 shows a decisional tree used to calculate the positivity of themethod in a patient with total blood PSA above 10 ng/ml.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

For purposes of clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the followingsubsections:

-   I. A Method to Assess the Presence of Prosate Cancer in a Sample by    Detecting PCA3 Nucleic Acid.-   II. Synthesis of Nucleic Acid.-   III. Probes and Primers.-   IV. A Kit for Detecting the Presence of PCA3 Nucleic Acid in a    Sample.    -   1. A Method to Assess the Presence of Prosate Cancer in a Sample        by Detecting PCA3 nucleic acid

The invention encompasses methods for detecting the presence of a PCA3nucleic acid together with a second prostate specific marker (e.g., PSA,hK2/KLK2, PSMA, transglutaminase 4, acid phophatase, PCGEM1) in abiological sample as well as methods for measuring the level of a PCA3nucleic acid in the sample. Such methods are useful for the diagnosticof prostate cancers associated with PCA3 overexpression.

The predisposition to develop prostate cancer or the presence of suchcancer may be detected based on the presence of an elevated amount ofPCA3 nucleic acid in a biological sample (e.g., urine) of a patient.Polynucleotides primers and probes may be used to detect the level ofPCA3 RNA present, which is indicative of the predisposition, presence orabsence of prostate cancer. In general the elevated amount of PCA3nucleic acid (e.g., PCA3 mRNA or fragments thereof) in a sample ascompared to the amount present in a normal control samples (or adetermined cut-off value) indicates that the sample contains prostatecancer or is susceptible to develop prostate cancer. In one embodiment,the detection of a second prostate-specific marker is also performed toserve as a control for the presence of prostate specific cells in thesample as well as to further validate the PCA3 detection results (e.g.,a negative result obtained with the detection of PCA3).

Of course, a number of different prostate specific marker can be used aslong as they can serve as a control for prostate RNA. Non-limitingexamples of such prostate-specific markers include PSA (SEQ ID NO 11)and other Kallikrein family members. In addition and as described above,markers such as hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase,PCGEM1 can also be used in accordance with the present invention.

One non limiting example of a method to detect PCA3 nucleic acid (e.g.PCA3 mRNA) in a biological sample is by (1) contacting a biologicalsample with at least one oligonucleotide probe or primer that hybridizesto a PCA3 polynucleotide; and (2) detecting in the biological sample alevel of oligonucleotide (i.e. probe(s) or primer(s)) that hybridizes tothe PCA3 polynucleotide. The sample is also tested for the presence ofsecond prostate-specific marker (e.g., PSA, hK2/KLK2, PSMA,transglutaminase 4, acid phosphatase, PCGEM1 mRNA or fragments thereof)to control for the presence of prostate cells in the sample (or theirnumber) as well as to further control a negative or positive resultobtained with the detection of PCA3. The second prostate specific markermay also be a prostate specific PCA3 RNA that is not associated withprostate cancer but is expressed in prostate cells. The amount of PCA3polynucleotide detected can be compared with a predetermined cut offvalue, and therefrom the predisposition, presence or absence of aprostate cancer in the patient is determined.

In a related aspect, the methods of the present invention can be usedfor monitoring the progression of prostate cancer in a patient. In thisparticular embodiment, the assays described above are performed overtime and the variation in the level of PCA3 nucleic acid and of anotherprostate specific marker (e.g., PSA mRNA) present in the sample (e.g.,urine sample) is evaluated. In general, prostate cancer is considered asprogressing when the relative (i.e. relatively to the amount of cells orcell components (e.g., protein or nucleic acids present therein) levelof PCA3 nucleic acid detected increases with time. In contrast a canceris not considered as progressing when the relative level of PCA3 nucleicacid either decreases or remains constant over time.

One skilled in the art can select the nucleic acid primers according totechniques known in the art as described above. Samples to be testedinclude but should not be limited to RNA samples from human tissue.

In a related aspect, it is possible to verify the efficiency of nucleicacid amplification and/or detection only, by performing external controlreaction(s) using highly purified control target nucleic acids added tothe amplification and/or detection reaction mixture. Alternatively, theefficiency of nucleic acid recovery from cells and/or organelles, thelevel of nucleic acid amplification and/or detection inhibition (ifpresent) can be verified and estimated by adding to each test samplecontrol cells or organelles (e.g., a define number of cells from aprostate cancer cell line expressing PCA3 and second marker) bycomparison with external control reaction(s). To verify the efficiencyof both, sample preparation and amplification and/or detection, suchexternal control reaction(s) may be performed using a reference testsample or a blank sample spiked with cells, organelles and/or viralparticles carrying the control nucleic acid sequence(s). For example, asignal from the internal control (IC) sequences present into the cells,viruses and/or organelles added to each test sample that is lower thanthe signal observed with the external control reaction(s) may beexplained by incomplete lysis and/or inhibition of the amplificationand/or detection processes for a given test sample. On the other hand, asignal from the IC sequences that is similar to the signal observed withthe external control reaction(s), would confirm that the samplepreparation including cell lysis is efficient and that there is nosignificant inhibition of the amplification and/or detection processesfor a given test sample. Alternatively, verification of the efficiencyof sample preparation only may be performed using external control(s)analyzed by methods other than nucleic acid testing (e.g. analysis usingmicroscopy, mass spectrometry or immunological assays).

Therefore, in one particular embodiment, the methods of the presentinvention uses purified nucleic acids, prostate cells or viral particlescontaining nucleic acid sequences serving as targets for an internalcontrol (IC) in nucleic acid test assays to verify the efficiency ofcell lysis and of sample preparation as well as the performance ofnucleic acid amplification and/or detection. More broadly, the IC servesto verify any chosen step of the process of the present invention.

IC in PCR or related amplification techniques can be highly purifiedplasmid DNA either supercoiled, or linearized by digestion with arestriction endonuclease and repurified. Supercoiled IC templates areamplified much less efficiently (about 100 fold) and in a lessreproducible manner than linearized and repurified IC nucleic acidtemplates. Consequently, IC controls for amplification and detection ofthe present invention are preferably performed with linearized andrepurified IC nucleic acid templates when such types of IC are used.

The nucleic acids, cells, and/or organelles are incorporated into eachtest sample at the appropriate concentration to obtain an efficient andreproducible amplification/detection of the IC, based on testing duringthe assay optimization. The optimal number of control cells added, whichis dependent on the assay, is preferentially the minimal number of cellswhich allows a highly reproducible IC detection signal without havingany significant detrimental effect on the amplification and/or detectionof the other genetic target(s) of the nucleic acid-based assay. A sampleto which is added the purified linearized nucleic acids, cells, viralparticles or organelles is generally referred to as a “spiked sample”.

Within certain embodiments, the amount of mRNA may be detected via aRT-PCR based assay. In RT-PCR, the polymerase chain reaction (PCR) isapplied in conjunction with reverse transcription. In such an assay, atleast two oligonucleotide primers may be used to amplify a portion ofPCA3 cDNA derived from a biological sample, wherein at least oneoligonucleotide is specific for (i.e. hybridizes to) a PCA3 RNA. Theamplified cDNA may then be separated and detected using techniques thatare well known in the art such as gel electrophoresis and ethidiumbromide staining. Amplification may be performed on biological samplestaken from a test patient and an individual who is not afflicted with aprostate cancer (control sample), or using other types of controlsamples. The amplification reaction may be performed on severaldilutions of cDNA (or directly on several dilutions of the biologicalsample) spanning, for example, two orders of magnitude. A value above apredetermined cut off value is indicative of the presence orpredisposition to develop prostate cancer. In general, the elevatedexpression of PCA3 nucleic acid in a biological sample as compared tocontrol samples indicates the presence or the predisposition to developprostate cancer.

In further embodiments, PCA3 RNA is detected in a nucleic acid extractfrom a biological sample by an in vitro RNA amplification method namedNucleic Acid Sequence-Based Amplification (NASBA). Other mRNAamplification methods well known in the art may also be used and includetranscriptase-mediated amplification (TMA), strand displacementamplification (SDA), the Qβ replicase system and Ligase chain reaction(LCR) (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and(Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi etal., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.Biol., 28:253-260; and Sambrook et al., 2000, Molecular Cloning—ALaboratory Manual, Third Edition, CSH Laboratories).

The amplification and/or detection of prostate cancer specific PCA3 RNAsequences and of the prostate specific marker can be carried outsimultaneously (e.g., multiplex real-time amplification assays.)

Alternatively, oligonucleotide probes that specifically hybridize understringent conditions to a PCA3 nucleic acid may be used in a nucleicacid hybridization assay (e.g., Southern and Northern blots, dot blot,slot blot, in situ hybridization and the like) to determine the presenceand/or amount of prostate cancer specific PCA3 polynucleotide in abiological sample.

Alternatively, oligonucleotides and primers could be designed todirectly sequence and assess the presence of prostate cancer specificPCA3 sequences in the patient sample following an amplification step.Such sequencing-based diagnostic methods are automatable and areencompassed by the present invention.

1. Synthesis of Nucleic Acid

The nucleic acid (e.g. DNA or RNA) for practicing the present inventionmay be obtained according to well known methods.

Isolated nucleic acid molecules of the present invention are meant toinclude those obtained by cloning as well as those chemicallysynthesized. Similarly, an oligomer which corresponds to the nucleicacid molecule, or to each of the divided fragments, can be synthesized.Such synthetic oligonucleotides can be prepared, for example, by thetriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185-3191(1981) or by using an automated DNA synthesizer.

An oligonucleotide can be derived synthetically or by cloning. Ifnecessary, the 5′-ends of the oligomers can be phosphorylated using T4polynucleotide kinase. Kinasing of single strands prior to annealing orfor labeling can be achieved using an excess of the enzyme. If kinasingis for the labeling of probe, the ATP can contain high specific activityradioisotopes. Then, the DNA oligomer can be subjected to annealing andligation with T4 ligase or the like. Of course the labeling of a nucleicacid sequence can be carried out by other methods known in the art.

II. Probes and Primers

The present invention relates to a nucleic acid for the specificdetection, in a sample, of the presence of PCA3 nucleic acid sequenceswhich are associated with prostate cancer, comprising theabove-described nucleic acid molecules or at least a fragment thereofwhich binds under stringent conditions to PCA3 nucleic acid.

In one preferred embodiment, the present invention relates to oligoswhich specifically target and enable amplification (i.e. primers) ofPCA3 RNA sequences associated with prostate cancer.

In another embodiment, PCA3 RNA can be detected using a specific probein an hybridization assay (e.g. Northern blot, dot blot, slot blot andthe like).

Oligonucleotide probes or primers of the present invention may be of anysuitable length, depending on the particular assay format and theparticular needs and targeted sequences employed. In a preferredembodiment, the oligonucleotide probes or primers are at least 10nucleotides in length (preferably, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 . . . ) and theymay be adapted to be especially suited for a chosen nucleic acidamplification system. Longer probes and primers are also within thescope of the present invention as well known in the art. Primers havingmore than 30, more than 40, more than 50 nucleotides and probes havingmore than 100, more than 200, more than 300, more than 500 more than 800and more than 1000 nucleotides in length are also covered by the presentinvention. Of course, longer primers have the disadvantage of being moreexpensive and thus, primers having between 12 and 30 nucleotides inlength are usually designed and used in the art. As well known in theart, probes ranging from 10 to more than 2000 nucleotides in length canbe used in the methods of the present invention. As for the % ofidentity described above, non-specifically described sizes of probes andprimers (e.g., 16, 17, 31, 24, 39, 350, 450, 550, 900, 1240 nucleotides,. . . ) are also within the scope of the present invention. In oneembodiment, the oligonucleotide probes or primers of the presentinvention specifically hybridize with a PCA3 RNA (or its complementarysequence). More preferably, the primers and probes will be chosen todetect a PCA3 RNA which is associated with prostate cancer. In oneembodiment, the probes and primers used in the present invention do nothybridize with the PCA3 gene (i.e. enable the distinction gene andexpressed PCA3). Other primers of the present invention are specific fora second prostate-specific marker such as PSA (SEQ ID NO 11). Of courseothervariants well known in the art can also be used (U.S. Pat. Nos.6,479,263 and 5,674,682) as second prostate specific marker. Because ofthe structural and sequence similarities of the PSA gene with othermembers of the kallikrein gene family, the appropriate selection of PSAsequences to serve as PSA-specific probes or primers is critical tomethods of amplification and/or detection of PSA specific nucleic acids.Examples of suitable primers for PSA, hK2/KLK2, PSMA, amplification anddetection (e.g., U.S. Pat. No. 6,551,778) are well known in the art aswell as for transglutaminase 4, acid phosphatase and PCGEM1. In oneembodiment, the PSA oligonucleotide may also hybridize to otherkallikrein family members such as kallikrein 2 (hK2/hKLK2). One exampleof such oligonucleotide is SEQ ID no 12.

As commonly known in the art, the oligonucleotide probes and primers canbe designed by taking into consideration the melting point ofhybridization thereof with its targeted sequence (see below and inSambrook et al., 1989, Molecular Cloning—A Laboratory Manual, 2ndEdition, CSH Laboratories; Ausubel et al., 1994, in Current Protocols inMolecular Biology, John Wiley & Sons Inc., N.Y.).

To enable hybridization to occur under the assay conditions of thepresent invention, oligonucleotide primers and probes should comprise anoligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%,74%), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%) and more preferably at least 90%(90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to aportion of a PCA3 polynucleotide. Probes and primers of the presentinvention are those that hybridize to PCA3 nucleic acid (e.g. cDNA ormRNA) sequence under stringent hybridization conditions and those thathybridize to PCA3 gene homologs under at least moderately stringentconditions. In certain embodiments probes and primers of the presentinvention have complete sequence identity to PCA3 gene sequence (e.g.cDNA or mRNA). However, probes and primers differing from the nativePCA3 gene sequence that keep the ability to hybridize to native PCA3gene sequence under stringent conditions may also be used in the presentinvention. It should be understood that other probes and primers couldbe easily designed and used in the present invention based on the PCA3nucleic acid sequence disclosed herein (SEQ ID NOs 9, 10 and 13) byusing methods of computer alignment and sequence analysis known in theart (cf. Molecular Cloning: A Laboratory Manual, Third Edition, editedby Cold Spring Harbor Laboratory, 2000).

For example, a primer can be designed so as to be complementary to ashort PCA3 RNA which is associated with a malignant state of theprostate cancer, whereas a long PCA3 RNA is associated with anon-malignant state (benign) thereof (PCT/CA00/01154 published under No.WO 01/23550). In accordance with the present invention, the use of sucha primer with the other necessary reagents would give rise to anamplification product only when a short PCA3 RNA (e.g., SEQ ID NO: 8)associated with prostate cancer is present in the sample. The longerPCA3 (e.g., SEQ ID NO: 7) would not give rise to an amplicon. Of course,the amplification could be designed so as to amplify a short and a longPCA3 mRNA. In such a format, the long PCA3 mRNA could be used as thesecond prostate specific marker.

In an embodiment as described above, the quantification of theamplification products of short versus long PCA3 could be carried outtogether with the detection of another prostate specific marker to serveas a molecular diagnostic test for prostate cancer. In anotherembodiment, primer pairs (or probes) specific for PCA3 could be designedto avoid the detection of the PCA3 gene or of unspliced PCA3 RNA. Forexample, the primers sequences to be used in the present invention couldspan two contiguous exons so that it cannot hybridize to an exon/intronjunction of the PCA3 gene. The amplification product obtained by the useof such primer would be intron less between two chosen exons (forexamples of such primers and probes see table 1 and 2 below). Therefore,unspliced variants and genomic DNA would not be amplified. It will berecognized by the person of ordinary skill that numerous probes can bedesigned and used in accordance with a number of embodiments of thepresent invention. Such tests can be adapted using the sequence of PCA3and that of the second prostate-specific marker. Of course, differentprimer pairs (and probes) can be designed from any part of the PCA3sequences (SEQ ID NOs: 7, 8, 9, 10 and 13) as well as from the sequenceof PSA (genbank accession number M27274, SEQ ID NO 11) or any otherchosen second prostate specific marker (e.g.,KLK2 (genbank acc. No.NM005551), PSMA (genbank acc. No.BC025672), transglutaminase 4 (genbankacc. No.BC007003), acid phosphatase (genbank acc. No. BC016344), PCGEM 1(genbank acc. No. AF223389)).

Probes of the invention can be utilized with naturally occurringsugar-phosphate backbones as well as modified backbones includingphosphorothioates, dithionates, alkyl phosphonates and α-nucleotides andthe like. Modified sugar-phosphate backbones are generally taught byMiller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987,Nucleic Acids Res., 14:5019. Probes of the invention can be constructedof either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), andpreferably of DNA.

Although the present invention is not specifically dependent on the useof a label for the detection of a particular nucleic acid sequence, sucha label might be beneficial, by increasing the sensitivity of thedetection. Furthermore, it enables automation. Probes can be labeledaccording to numerous well-known methods (Sambrook et al., 2000, supra).Non-limiting examples of detectable markers and labels include ³H, ¹⁴C,³²P, and ³⁵S, ligands, fluorophores, chemiluminescent agents, enzymes,and antibodies. Other detectable markers for use with probes, which canenable an increase in sensitivity of the method of the invention,include biotin and radionucleotides. It will become evident to theperson of ordinary skill that the choice of a particular label dictatesthe manner in which it is bound to the probe.

As commonly known, radioactive nucleotides can be incorporated intoprobes of the invention by several methods. Non-limiting examplesthereof include kinasing the 5′ ends of the probes using gamma ³²P ATPand polynucleotide kinase, using the Klenow fragment of Pol I of E. coliin the presence of radioactive dNTP (e.g. uniformly labeled DNA probeusing random oligonucleotide primers), using the SP6/T7 system totranscribe a DNA segment in the presence of one or more radioactive NTP,and the like.

In one embodiment, the label used in a homogenous detection assay is achemiluminescent compound (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737 and5,639,604), more preferably an acridinium ester (“AE”) compound, such asstandard AE or derivatives thereof. Methods of attaching labels tonucleic acids and detecting labels-are well known (e.g., see Sambrook etal., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Habor, N.Y., 1989), Chapt. 10; U.S. Pat.Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174 and 4,581,333; andEuropean Pat. App. No.0 747 706). Preferred methods of labeling a probewith an AE compound attached via a linker have been previously describeddetail (e.g., see U.S. Pat. No. 5,639,604, Example 8).

Amplification of a selected, or target, nucleic acid sequence may becarried out by a number of suitable methods. See generally Kwoh et al.,1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniqueshave been described and can be readily adapted to suit particular needsof a person of ordinary skill. Non-limiting examples of amplificationtechniques include polymerase chain reaction (PCR, RT PCR . . . ),ligase chain reaction (LCR), strand displacement amplification (SDA),transcription-based amplification, the Qβ replicase system and NASBA(Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi etal., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.Biol., 28:253-260; and Sambrook et al., 2000, supra). Other non-limitingexamples of amplification methods include rolling circle amplification(RCA); signal mediated amplification of RNA technology (SMART); splitcomplex amplification reaction (SCAR); split promoter amplification ofRNA (SPAR).

Non-limiting examples of suitable methods to detect the presence of theamplified products include the followings: agarose or polyacrylamidegel, addition of DNA labeling dye in the amplification reaction (such asethidium bromide, picogreen, SYBER green, etc.) and detection withsuitable apparatus (fluorometer in most cases). Other suitable methodsinclude sequencing reaction (either manual or automated); restrictionanalysis (provided restriction sites were built into the amplifiedsequences), or any method involving hybridization with a sequencespecific probe (Southern or Northern blot, TaqMan™ probes, molecularbeacons, and the like). Of course, other amplification methods areencompassed by the present invention. Molecular beacons are exemplifiedherein as one method for detecting the amplified products according tothe present invention (see below).

Of course in some embodiment direct detection (e.g., sequencing) of PCA3cancer specific sequences as well as that of another prostate specificmarker in a sample may be performed using specific probes or primers.

In one embodiment, the present invention has taken advantage oftechnological advances in methods for detecting and identifying nucleicacids. Therefore, the present invention is suitable for detection by oneof these tools called molecular beacons.

Molecular beacons are single-stranded oligonucleotide hybridizationprobes/primers that form a stem loop structure. The loop contains aprobe sequence that is complementary to a target sequence, and the stemis formed by the annealing of complementary arm sequences that arelocated on either side of the probe/primer sequence. A fluorophore iscovalently linked to the end of one arm and a quencher is covalentlylinked to the end of the other arm. Molecular beacons do not fluorescewhen they are free in solution. However, when they hybridize to anucleic acid strand containing a target sequence they undergocomformational change that enables them to fluoresce brightly (see U.S.Pat. Nos. 5,925,517, and 6,037,130). Molecular beacons can be used asamplicon detector probes/primers in diagnostic assays. Becausenonhybridized molecular beacons are dark, it is not necessary to isolatethe probe-target hybrids to determine for example, the number ofamplicons synthesized during an assay. Therefore, molecular beaconssimplify the manipulations that are often required when traditionaldetection and identifications means are used.

By using different colored fluorophores, molecular beacons can also beused in multiplex amplification assays such as assays that target thesimultaneous amplification and detection of PCA3 nucleic acid and of thesecond specific prostate nucleic acid (e.g., PSA, hK2/KLK2, PSMA,transglutaminase 4, acid phosphatase and PCGEM1). The design ofmolecular beacons probes/primers is well known in the art and softwaresdedicated to help their design are commercially available (e.g., Beacondesigner from Premier Biosoft International). Molecular beaconprobes/primers can be used in a variety of hybridization andamplification assays (e.g., NASBA and PCR).

In accordance with one embodiment of the present invention, theamplified product can either be directly detected using molecularbeacons as primers for the amplification assay (e.g., real-timemultiplex NASBA or PCR assays) or indirectly using, internal to theprimer pair binding sites, a molecular beacon probe of 18 to 25nucleotides long (e.g., 18, 19, 20, 21, 22, 23, 24, 25) wichspecifically hybridizes to the amplification product. Molecular beaconsprobes or primers having a length comprised between 18 and 25nucleotides are preferred when used according to the present invention(Tyagi et al., 1996, Nature Biotechnol. 14: 303-308). Shorter fragmentscould result in a less fluorescent signal, whereas longer fragmentsoften do not increase significantly the signal. Of course shorter orlonger probes and primers could nevertheless be used.

Examples of nucleic acid primers which can be derived from PCA3 RNAsequences are shown hereinbelow in Table 1: TABLE 1 NUCLEIC ACID PRIMERSSize (no. of bases) Nucleotides Exon 1 98 1-98 of SEQ ID NO: 9 Exon 2165 99-263 of SEQ ID NO: 9 Exon 3 183 264-446 of SEQ ID NO: 9 Exon 4a539 447-985 of SEQ ID NO: 9 Exon 4b 1052 986-2037 of SEQ ID NO: 9 Exon 1120 1-120 of SEQ ID NO: 10 Exon 2 165 121-285 of SEQ ID NO: 10 Exon 3183 286-468 of SEQ ID NO: 10 Exon 4a 539 469-1007 of SEQ ID NO: 10 Exon4b 1059 1008-2066 of SEQ ID NO: 10 Exon 4c 556 2067-2622 of SEQ ID NO:10 Exon 4d 960 2623-3582 of SEQ ID NO: 10 Exon junction 1 20 89-108 ofSEQ ID NO: 9 Exon junction 1 20 109-128 of SEQ ID NO: 10 Exon junction 220 252-271 of SEQ ID NO: 9 Exon junction 2 20 274-293 of SEQ ID NO: 10Exon junction 3 20 435-454 of SEQ ID NO: 9 Exon junction 3 20 457-476 ofSEQ ID NO: 10 Exon junction 4 20 974-993 of SEQ ID NO: 9 Exon junction 420 996-1015 of SEQ ID NO: 10 Exon junction 5 20 2055-2074 of SEQ ID NO:10 Exon junction 6 20 2611-2630 of SEQ ID NO: 10

It should be understood that the sequences and sizes of the primerstaught in Table 1 are arbitrary and that a multitude of other sequencescan be designed and used in accordance with the present invention.

While the present invention can be carried out without the use of aprobe which targets PCA3 sequences, such as the exon junctions of PCA3in accordance with the present invention, such probes can add a furtherspecificity to the methods and kits of the present invention. Examplesof specific nucleic acid probes which can be used in the presentinvention (and designed based on the exonic sequences shown in Table 1)are set forth in Table 2, below: TABLE 2 NUCLEIC ACID PROBES Size (no.of bases) Nucleotides Probe 1 20 1-20 of SEQ ID NO: 9 Probe 2 30 1-30 ofSEQ ID NO: 9 Probe 3 40 1-40 of SEQ ID NO: 9 Probe 4 20 1-20 of SEQ IDNO: 10 Probe 5 30 1-30 of SEQ ID NO: 10 Probe 6 40 1-40 of SEQ ID NO: 10Probe 7 20 89-108 of SEQ ID NO: 9 Probe 8 30 114-143 of SEQ ID NO: 10Probe 9 30 257-286 of SEQ ID NO: 9 Probe 10 20 284-303 of SEQ ID NO: 10Probe 11 20 274-293 of SEQ ID NO: 9

Of course, as will be understood by the person of ordinary skill, amultitude of additional probes can be designed from the same or otherregion of SEQ ID NO. 9 as well as from SEQ ID NO. 10 and 13 and othersequences of the present invention, whether they target exon junctionsor not. It will be clear that the sizes of the probes taught in Table 2are arbitrary and that a multitude of other sequences can be designedand used in accordance with the present invention.

It will be readily recognized by the person of ordinary skill, that thenucleic acid sequences of the present invention (e.g., probes andprimers) can be incorporated into anyone of numerous established kitformats which are well known in the art.

In one embodiment of the above-described method, a nucleic acid probe isimmobilized on a solid support. Examples of such solid supports include,but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, and acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling nucleic acidprobes to such solid supports are well known in the art.

The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids (e.g., urine). The sample used in theabove-described methods will vary based on the assay format, thedetection method and the nature of the tissues, cells or extracts to beassayed. Methods for preparing nucleic acid extracts of cells are wellknown in the art and can be readily adapted in order to obtain a samplewhich is compatible with the method utilized. Preferably the sample is aurine sample. When the urine sample is used, it should contain at leastone prostate cell in order to enable the identification of the prostatespecific marker of the present invention. In fact, assuming that thehalf-life of PCA3 mRNA in an untreated biological sample is not suitablefor easily enabling the preservation of the integrity of its sequence,the collected sample, whether urine or otherwise, should, prior to atreatment thereof contain at least one prostate cell. It will berecognized that the number of cells in the sample will have an impact onthe validation of the test and on the relative level of measured PCA3(or second prostate specific marker).

III. A Kit for Detecting the Presence of PCA3 Nucleic Acid in a Sample

In another embodiment, the present invention relates to a kit fordiagnosing prostate cancer in a manner which is both sensitive andspecific (i.e lowering the number of false positives). Such kitgenerally comprises a first container means having disposed therein atleast one oligonucleotide probe or primer that hybridizes to a prostatecancer-specific PCA3 nucleic acid sequence. In one embodiment, thepresent invention also relates to a kit further comprising in a secondcontainer means oligonucleotide probes or primers which are specific toa second prostate specific marker, thereby validating a negative resultwith PCA3.

In a particular embodiment of the present invention, this kit (K)comprises a primer pair which enables the amplification of PSA,hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase and PCGEM1) Ofcourse the present invention also encompasses the use of a thirdprostate specific marker.

Oligonucleotides (probes or primers) of the kit may be used, forexample, within a NASBA, PCR or hybridization assay. Amplificationassays may be adapted for real time detection of multiple amplificationproducts (i.e. multiplex real time amplification assays).

In a related particular embodiment, the kit further includes othercontainers comprising additional components such as additionaloligonucleotide or primer and/or one or more of the following: buffers,reagents to be used in the assay (e.g. wash reagents, polymerases orelse) and reagents capable of detecting the presence of bound nucleicacid probe or primers. Examples of detection reagents include, but arenot limited to radiolabelled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), and affinity labeled probes(biotin, avidin, or steptavidin). In one embodiment, the detectionreagents are molecular beacon probes which specifically hybridizes tothe amplification products. In another embodiment, the detectionreagents are chemiluminescent compounds such as Acridinium Ester (AE).

For example, a compartmentalized kit in accordance with the presentinvention includes any kit in which reagents are contained in separatecontainers. Such containers include small glass containers, plasticcontainers or strips of plastic or paper. Such containers allow theefficient transfer of reagents from one compartment to anothercompartment such that the samples and reagents are not crosscontaminated and the agents or solutions of each container can be addedin a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample(e.g., an RNA extract from a biological sample or cells), a containerwhich contains the primers used in the assay, containers which containenzymes, containers which contain wash reagents, and containers whichcontain the reagents used to detect the extension products. As mentionedabove, the separation or combination of reagents can be adapted by theperson of ordinary skill to which this invention pertain, according tothe type of kit which is preferred (e.g., a diagnostic kit based onamplification or hybridization methods or both), the types of reagentsused and their stability or other intrinsic properties. In oneembodiment, one container contains the amplification reagents and aseparate container contains the detection reagent. In anotherembodiment, amplification and detection reagents are contained in thesame container.

Kits may also contain oligonucleotides that serve as capture oligomersfor purifying the target nucleic acids from a sample. Examples ofcapture oligomers have sequences of at least 15 nucleotidescomplementary to a portion of the PCA3 target nucleic acid. Embodimentsof capture oligomers may have additional bases attached to a 3′ or 5′end the sequence that is complementary to the PCA3 target sequence whichmay act functionally in a hybridization step for capturing the targetnucleic acid. Such additional sequences are preferably a homopolymerictail sequence, such as a poly-A or poly-T sequence, although otherembodiments of tail sequences are included in capture oligomers of thepresent invention. In one embodiment, CAP binding protein (e.g.,eIF4G-4E) or part thereof may be used to capture cap-structurecontaining mRNAs (Edery et al., 1987, Gene 74(2): 517-525). In anotherembodiment, a non specific capture reagent is used (e.g., silica beads).

Kits useful for practicing the methods of the present invention mayinclude those that include any of the amplification oligonucleotidesand/or detection probes disclosed herein which are packaged incombination with each other. Kits may also include capture oligomers forpurifying the PCA3 target nucleic acid from a sample, which captureoligomers may be packaged in combination with the amplificationoligonucleotides and/or detection probes.

In a further embodiment, cells contained in voided urine samplesobtained after an attentive digital rectal examination are harvested andlysed in a lysis buffer. Nucleic acids are extracted (e.g., from thelysate by solid phase extraction on silica beads for example). Detectionof the presence of RNA encoded by the PCA3 gene in the nucleic acidextract is done by an in vitro specific RNA amplification coupled toreal-time detection of amplified products byfluorescent specific probes.In this method, simultaneously to the amplification of the PCA3 prostatecancer specific RNA undergoes the amplification of the secondprostate-specific marker (such as the PSA RNA) as a control for thepresence in the urine sample of prostate cells.

The screening and diagnostic methods of the invention do not requirethat the entire PCA3 RNA sequence be detected. Rather, it is onlynecessary to detect a fragment or length of nucleic acid that issufficient to detect the presence of the PCA3 nucleic acid from a normalor affected individual, the absence of such nucleic acid, or an alteredstructure of such nucleic acid (such as an aberrant splicing pattern).For this purpose, any of the probes or primers as described above areused, and many more can be designed as conventionally known in the artbased on the sequences described herein and others known in the art.

It is to be understood that although the following discussion isspecifically directed to human patients, the teachings are alsoapplicable to any animal that expresses PCA3.

The diagnostic and screening methods of the invention are especiallyuseful for a patient suspected of being at risk for developing a diseaseassociated with an altered expression level of PCA3 based on familyhistory, or a patient in which it is desired to diagnose a PCA3-relateddisease (ex. prostate cancer). The method of the present invention mayalso be used to monitor the progression of prostate cancer in patient asdescribed above.

The present invention is illustrated in further details by the followingnon-limiting example. The examples are provided for illustration onlyand should not be construed as limiting the scope of the invention.

EXAMPLE 1 CLINICAL PERFORMANCE USING ONE ILLUSTRATIVE EMBODIMENT OF THEMETHODS OF THE PRESENT INVENTION

To estimate the clinical performance of the method, a pilot study wasdone on 517 patients planned to undergo ultrasound guided needlebiopsies coming from five university medical centers located in Montrealand Quebec (Canada) between September 2001 and June 2002. Each samplewas processed using the following steps:

Sample Collection

Following an attentive digital rectal examination, the first 20 to 30 mlof voided urine was collected in sterile 80 ml plastic containers(patient urinates directly in the sterile container).

An equal volume of Sample buffer (0.1M phosphate (0.06M Na₂HPO₄, 0.04MNaH₂PO₄) 0.3M NaCl, pH 7.0,) was immediately added and the solutionmixed by inversion.

If not processed immediately, samples were refrigerated between 2-8° C.for up to three days until further processing. In view of the cellrecovery step, freezing should be avoided.

Cell Recovery

The sample was mixed by inversion; and the container gently tapped onthe counter in order to detach cells from the inner walls thereof. Thesample was then transferred into one or two (if necessary) conicalpolypropylene tubes (40 ml/tube).

The cells were pelleted by centrifugation in a tabletop centrifuge at1400 g for 15 minutes. Finally, the supernatant was decanted and thecells were immediately lysed.

Cell Lysis

400 μl of Lysis Buffer (4.68M GUSCN, 20 mM EDTA, 1.2% Triton X-100™, 46mM Tris-HCl, pH 7.2) was added to the urine cell pellet.

The cell pellet was then vigorously vortexed for 20 seconds in order tolyse the cells. It is important to make sure that no particulate matteris left. The lysate was transparent and not too viscous.

The lysate was transferred into a 1.5 ml microtubes and vortexed for 30seconds.

If desired, the lysed cells can now be stored at ≦−70° C. indefinitely.

Nucleic Acid Extraction

The silica suspension (60 g silica type ±80% particle size 1-5 μm, addMilliQ water at a final volume of 500 ml) was first vigorously vortexedfor 30 seconds until an opaque homogeneous suspension was obtained.

200 μl of the suspension was then immediately removed and added to thelysed specimen. All tubes were subsequently vigorously vortexed for 15seconds to bind nucleic acids to the silica.

On a test tube rack, a series of Microspin™ Columns identifying eachfilter unit with the appropriate number of patient were prepared.

The content of each microtube containing the lysed cells and the silicawere transferred into the membrane filter unit of one Microspin™ Column.To facilitate the transfer of the particulate matter, the microtube wasvortexed briefly (approximately 5 seconds) in order to resuspend thecontent. The same was done before transferring. Tips were changedbetween samples.

The Microspin™ columns were centrifuged in a non-refrigeratedmicrocentrifuge at 10,000 RPM for 5 minutes at room temperature (18°C.-25° C.). The membrane filter retained silica-bound nucleic acidswhereas other cellular components remained in the flow-through.

Meanwhile, a series of 2 ml microtubes corresponding to the number ofMicrospin™ columns were prepared.

The membrane filter units containing the silica were transferred to new2 mL microtubes. 500 μl of Wash Buffer (5.3M GuSCN, 52 mM Tris-HCl pH6.4) was added to each membrane filter unit. The Microspin™ columns werethen centrifuged in a non-refrigerated microfuge at 10,000 RPM for 5minutes at room temperature.

On a test tube rack, a new series of 2 ml microtubes were prepared.

The membrane filter units with the silica were transferred to the new 2ml microtubes. 600 μl ethanol 70% was added to the membrane filterunits. The Microspin™ columns were then centrifuged in anon-refrigerated microfuge at 10,000 RPM for 5 minutes at roomtemperature.

On a test tube rack, a new series of 2 ml microtubes were prepared.

The membrane filter units with the silica are transferred to a new 2 mlmicrotube. Discard the microtubes containing the flow-through.

The membrane filter unit containing microtubes were then transferred toa heating block at 65° C.±1° C. installed under a fume hood.

All tubes were opened carefully to ensure evaporation, and incubated forapproximately 10 minutes to dry the silica.

200 μl Elution Buffer (Dnase/Rnase-free water) to each membrane filterunit was added.

The membrane filter units were then centrifuged in a microfuge at 10,000RPM for 5 minutes at room temperature.

The elution steps were repeated once to obtain a second eluate. Thesesteps elute nucleic acids from the silica and concentrate them in theflow-through.

The microfilter units were disposed and the two microtubes containingthe nucleic acid elution were kept.

For each eluate, three aliquots of ≅50 μl of nucleic acids were storedat ≦−70° C.

In Vitro RNA Amplification and Detection

The nucleic acid eluate sample to test was first thawed on ice. Thereaction mix was then prepared according to the number of reactions tobe performed. Each sample was made at least in duplicate.

10 μl of the reaction mix was distributed in identified microtubes [80mM Tris-HCl pH 8.5, 24 mM MgCl₂, 180 mM KCl, 10 mM DTT, 2 mM of eachdNTP, 4 mM of rATP, rUTP, CTP, 3 mM rGTP, 1 mM ITP, 30% DMSO, 3%sucrose, 1% D-Mannitol, 1% Dextran T-40, 208 nM PSA primers (N2psaP1B,SEQ. ID NO 1 and N2psaP2B, SEQ. ID NO 2), 417 nM PCA-3 primers(N0pcaP1A, SEQ. ID NO 3 and N0pcaP2B, SEQ. ID NO 4), 84 nM PSA beacon(BpsaRD-4, SEQ. ID NO 5), 166 nM PCA-3 beacon (BpcaFD-4, SEQ. ID NO 6).

5 μl of nucleic acid sample eluate was added in each tube and mixed.

Tubes were placed in a thermocycler™, heated at 65° C.±1° C. for aperiod of 5 minutes and then the temperature was kept at 41° C. After 5minutes at 41° C., tubes were retrieved and centrifuged briefly in orderto remove the condensation drops from the lids.

The next steps were better carried out quickly, and the tube temperaturewas preferably kept at 41° C.

5 μl of the enzyme mix (375 mM sorbitol, 0.105 μg/μl BSA, 0.08 units ofRnaseH, 32.0 units of T7 RNA polymerase, 6.4 units of AMV-RT) was thenquickly added to each tube and the tubes were gently mixed.

The tubes were put back into the EasyQ™ incubator. When the last tubewas in place, the incubator was kept at a temperature of 41° C±0.5° C.for 5 minutes.

The tubes were then briefly centrifuged. Quickly, all tubes weretransferred in a thermostated spectrofluorimeter for in vitro RNAamplification and real time amplified product detection with thefollowing characteristics: (1) the light source was a quartz-halogenlamp, (2) the filter used for ROX (6-carboxy-x-rhodamine N-succinimidylester) fluorescence was at 550-620 nm and for FAM (6-carboxyfluoresceinN-hydroxysuccinimide ester) was at 485-530 nm, (3) the fluorescenceintegration time per tube was 20 msec; and (4) ROX and FAM emission wasread each 30 sec and the tube block was set at the temperature of 41°C.±1° C.

Results

Fluorescence data generated during the two hours of amplificationunderwent fitting following the approach of Brown [Computer Methods andPrograms in Biomedicine 65 (2001) 191-200].

Based on the PSA ratio (fluo maxlfluo min) cut off of 1.3, out of the517 patients who have been tested, 443 had adequate quantities ofprostate cells in the urine.

In this population of patients, 34% (151/443) had prostate cancerconfirmed by histology. TABLE 3 Positive Biopsies versus tPSA CategoriestPSA Percentage of Patients Positive Biopsies <4 ng/ml 21% (n = 94) 20%(n = 19) 4-10 ng/ml 55% (n = 243) 35% (n = 85) >10 ng/ml 24% (n = 106)44% (n = 47)

Clinical specificity (Sp) and sensitivity (Se) of the method has beenestimated following a tree-structured classification using S-plus™software [Insightful Corporation, Seattle, Wash., USA] starting from rawfluorimeter data. Three structured trees have been defined for the threetypes of patients defined as having a total blood PSA (tPSA) below 4ng/ml, between 4-10 ng/ml and above 10 ng/ml (see FIGS. 2-4 and TABLE 4.TABLE 4 Method sensitivity and specificity tPSA Number Se % Sp % <4ng/ml 94 74 (14/19) 91 (68/75) 4-10 ng/ml 243 59 (50/85) 91(144/158) >10 ng/ml 106 79 (37/47) 80 (47/59) Overall 443 67 (101/151)89 (259/292)

TABLE 5 Method performance versus total tPSA and free fPSA Se % Sp %tPSA ≧ 2.5 ng/ml 100% (58/58) 6% (5/88) tPSA ≧ 4.0 ng/ml 88% (51/58) 15%(13/88) FPSA/tPSA ≦ 0.15 72% (42/58) 56% (49/88) FPSA/tPSA ≦ 0.13 66%(38/58) 67% (59/88) uPM3 ™ 64% (37/58) 91% (80/88)146/443 patients with available fPSA

The study demonstrated that the method has a positive predictive value(PPV) of 75%, compared to total PSA (>4.0 ng/ml) with a PPV of only 38%.The negative predictive value of the method is 84%, compared to 81% fortPSA. The overall accuracy of the method is 81%, compared with anaccuracy of 47% for tPSA.

Although the present invention has been described hereinabove by way ofillustrative embodiments thereof, it will be appreciated by one skilledin the art from reading of this disclosure that various changes in formand detail can be made without departing from the spirit and nature ofthe invention as defined in the appended claims. For example, variousother amplification assays or detection assays, different probes andprimers sequences as well as slightly different temperature and time ofincubation may be used according to the present invention.

1. A method for determining a predisposition, or presence of prostatecancer in a patient comprising: a) contacting a biological sample ofsaid patient with at least one oligonucleotide that hybridizes to a PCA3polynucleotide selected from the group consisting of: i) apolynucleotides according to SEQ ID NOs 9, 10 and 13; ii) apolynucleotide sequence that hybridizes under high stringency conditionsto the polynucleotide sequence in i); and iii) a polynucleotide sequencefully complementary to i) or ii); and contacting said biological samplewith at least one oligonucleotide that hybridizes with a second prostatespecific polynucleotide, b) detecting in said biological sample anamount of PCA3 and second prostate specific polynucleotides; andcomparing the amount of PCA3 polynucleotide that hybridizes to theoligonucleotide to a predetermined cut off value, and therefromdetermining the presence or absence of prostate cancer in the biologicalsample.
 2. The method of claim 1, wherein said second specific prostatespecific nucleic acid is selected from the group consisting of: PSA,human kallikrein 2, PSMA, transglutaminase 4, acid phosphatase andPCGEM1 nucleic acid.
 3. The method of claim 2, wherein said prostatespecific nucleic acid is PSA.
 4. The method of claim 3, wherein said PSAsequence hybridizes to human kallikrein
 2. 5. The method of claim 1,wherein the amount of PCA3 polynucleotide and of the second specificprostate cancer polynucleotide is determined using an assay selectedfrom the group consisting of: a) an amplification assay; and b) ahybridization assay.
 6. The method of claim 5, wherein saidamplification assay is an in vitro RNA amplification method.
 7. Themethod of claim 6, wherein said RNA amplification method is selectedfrom the group consisting of: a) nucleic acid sequence-basedamplification (NASBA); b) polymerase chain reaction (PCR); c)transcription mediated amplification assay (TMA); and d) ligase chainreaction.
 8. The method of claim 6, wherein said amplification of PCA3and said second prostate specific nucleic acid is performedsimultaneously.
 9. The method of claim 6, wherein said amplification ofPCA3 is carried out using a primer pair composed of SEQ ID NOs: 3 and 4.10. The method of claim 6, wherein said detection is performed byfluorescence, chimiluminescence or colorimetry detection.
 11. The methodof claim 10, wherein said detection of PCA3 is carried out usingacridinium ester compounds.
 12. The method of claim 6, wherein saiddetection of PCA3 is carried out using a molecular beacon.
 13. Themethod of claim 12, wherein said beacon has the sequence set forth inSEQ ID NO:
 6. 14. The method of one of claims 6, wherein said secondprostate specific nucleic acid is PSA and said amplification thereof iscarried out using a primer pair composed of SEQ ID NOs: 1 and
 2. 15. Themethod of claim 14, wherein said detection of PSA is carried out usingacridinium ester compounds.
 16. The method of claim 14, wherein saiddetection of PSA is carried out using a PSA molecular beacon.
 17. Themethod of claim 16, wherein said PSA beacon has the sequence set forthin SEQ ID NO:
 5. 18. The method of claim 1, wherein said sample containsat least one prostate cell and said at least one cell is collected fromsaid sample prior to step a).
 19. The method of claim 18, wherein saidnucleic acid is extracted from said at least one prostate cell.
 20. Themethod of claim 19, wherein said nucleic acid is RNA.
 21. The method ofclaim 20, wherein said RNA is extracted using a silica-based method. 22.The method of claims 1, wherein said sample is selected from the groupconsisting of: a) urine; b) blood or fraction thereof; and c) prostatebiopsy.
 23. The method of claim 22 wherein said sample is urine.
 24. Themethod of claim 23, wherein said urine is collected following a digitalrectal examination, thereby increasing the number of prostate cells insaid sample.
 25. The method of claim 1, further comprising: c) repeatingsteps (a) and (b) using a biological sample from the patient at asubsequent point in time; and d) comparing the relative amount of saidPCA3 polynucleotide detected in step (c) to the relative amount of PCA3polynucleotide detected in step (b) and therefrom monitoring theprogression of the prostate cancer in the patient.
 26. The method ofclaim 1, wherein the detection of the second prostate specificpolynucleotide validates a negative result for PCA3 detection.
 27. Themethods of claim 1, wherein the biological sample is spiked with aninternal control IC selected from the group consisting of: a) purifiednucleic acid; b) cells; c) viral particules containg target nucleicacids; and d) organelles.
 28. The method of claim 6, wherein RNA isextracted using a target capture method.
 29. The method of claim 1,wherein said detection of PCA3 is carried out using chemiluminescentlabels in a homogenous detection method.
 30. A diagnostic kit for thedetection of prostate cancer or the risk of developing same in a patientcomprising: a) at least one container having disposed therein at leastone oligonucleotide probe or primer that hybridizes to one of: i) a PCA3nucleic acid sequence according to SEQ ID NO: 9, 10 and 13; ii) asequence which is fully complementary to i); and iii) a sequence whichhybridizes under high stringency conditions to i) or ii); b) at leastone oligonucleotide probe or primer that hybridizes with a secondprostate specific nucleic acid or complement thereof; and c) reagentsenabling a detection of PCA3 and of said second prostate specificnucleic acid when said PCA3 or second prostate-specific nucleic acidsequence is present.
 31. The diagnostic kit according to claim 30,wherein the detection reagent comprises a reporter group or labelselected from the group consisting of: a) radioisotopes; b) enzymes; c)fluorescent groups; d) biotin; e) chemiluminescent groups; and f) dyeparticles.
 32. The kit of claim 30, wherein said PCA3 nucleic acid andsaid second prostate specific nucleic acid are amplified simultaneouslyin the same container.
 33. The kit of claim 30, wherein the detection ofsaid PCA3 nucleic acid and said second prostate specific nucleic acid isperformed in the same container.
 34. The kit of claim 30, furthercomprising an internal control (IC) as well as a primer, and/or probe,and/or reagent for the amplification, and/or hybridization, and/ordetection of said internal control.
 35. The kit of claim 34, whereinsaid IC is selected from the group consisting of: a) purified nucleicacid; b) cells; c) viral particules containg target nucleic acids; andd) organelles.
 36. A kit for assessing the presence of prostate canceror the risk of developing same in a patient comprising: a) a firstprimer pair specific for amplifying a PCA3 nucleic acid associated withprostate cancer present in a patient sample; b) a second primer pairspecific for amplifying a second prostate-specific nucleic acid; and c)reagents enabling a detection of PCA3 and of said second prostatespecific nucleic acid amplification products when said PCA3 or secondprostate-specific nucleic acid sequence is present.
 37. A method fordetecting prostate cancer in a human patient, comprising: a) performingan in vitro nucleic acid amplification assay on a biological sample ofsaid patient or extract thereof using a first primer pair which isspecific to a prostate cancer specific PCA3 sequence and a second primerpair which is specific to a prostate specific nucleic acid sequence; andb) detecting said PCA3 sequence and said prostate specific nucleic acidsequence, wherein, a detection of said PCA3 nucleic acid sequence or alevel thereof correlates with a risk of developing prostate cancer or toa presence of prostate cancer in said patient, and wherein an absence ofdetection of said PCA3 nucleic acid sequence or lower level thereof insaid sample validates an absence of prostate cancer or a lower risk ofdeveloping same, when said second prostate specific nucleic acid isdetected.
 38. The method of one of claim 1, wherein said nucleic acidamplification is carried-out in real time.
 39. The method of claim 37,wherein said detection is performed by fluorescence, chimiluminescenceor colorimetry detection.
 40. The method of claim 8, wherein saidamplification of PCA3 and said second prostate specific nucleic acid isperformed simultaneously in one container.